CN115925514B - Method for purifying cembratriene diol in tobacco leaves by multi-dimensional preparation liquid chromatography - Google Patents
Method for purifying cembratriene diol in tobacco leaves by multi-dimensional preparation liquid chromatography Download PDFInfo
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- 241000208125 Nicotiana Species 0.000 title claims abstract description 39
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 35
- 238000004811 liquid chromatography Methods 0.000 title claims abstract description 27
- 150000002009 diols Chemical class 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims abstract description 44
- 239000007788 liquid Substances 0.000 claims abstract description 29
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000002386 leaching Methods 0.000 claims abstract description 15
- 239000002904 solvent Substances 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 9
- 239000000284 extract Substances 0.000 claims abstract description 5
- 238000010828 elution Methods 0.000 claims description 41
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical group O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 16
- 238000000746 purification Methods 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- AINBZKYUNWUTRE-UHFFFAOYSA-N ethanol;propan-2-ol Chemical group CCO.CC(C)O AINBZKYUNWUTRE-UHFFFAOYSA-N 0.000 claims description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical group CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 5
- 239000000945 filler Substances 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 5
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- 239000000126 substance Substances 0.000 abstract description 6
- 238000013375 chromatographic separation Methods 0.000 abstract description 4
- 150000001875 compounds Chemical class 0.000 abstract description 2
- 238000000148 multi-dimensional chromatography Methods 0.000 abstract description 2
- 239000002699 waste material Substances 0.000 description 15
- 238000000926 separation method Methods 0.000 description 11
- 238000011068 loading method Methods 0.000 description 10
- 238000004587 chromatography analysis Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
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- 239000012535 impurity Substances 0.000 description 4
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- 239000000287 crude extract Substances 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
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- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 1
- LHORCXXUZJAMPU-UHFFFAOYSA-N 1,7,11-trimethyl-4-(propan-2-yl)cyclotetradecane Chemical compound CC(C)C1CCC(C)CCCC(C)CCCC(C)CC1 LHORCXXUZJAMPU-UHFFFAOYSA-N 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
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- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to the field of chemical preparation, in particular to a method for purifying cembratriene glycol in tobacco leaves by multi-dimensional preparation liquid chromatography, which comprises the following steps: a1 Adding a solvent into the tobacco leaf sample for leaching to obtain an extracting solution; a2 Purifying and eluting the extract obtained in the step A1) by a first dimension liquid chromatographic column, purifying and eluting by a second dimension liquid chromatographic column, capturing and eluting by a capturing column array, and then performing third dimension liquid chromatographic column fraction and eluting to provide purified alpha-cembratriene glycol and beta-cembratriene glycol. The invention uses multidimensional chromatography to separate the cembratriene diol in the tobacco with multi-dimension and high efficiency. The cembratriene diol is a compound with relatively poor thermal stability, and the multidimensional chromatographic separation process is not contacted with the external environment, so that the cembratriene diol is more suitable for separating substances with poor thermal stability; the detection of multiple detectors eliminates interference and results are reliable.
Description
Technical Field
The invention relates to the field of chemical preparation, in particular to a method for purifying cembratriene glycol in tobacco leaves by multi-dimensional preparation liquid chromatography.
Background
The cembratriene diol is used as an important tobacco flavor precursor substance, is an important precursor substance of cigarette aroma components, and has various biological activities of inhibiting tumor cell growth, prostaglandin synthesis, nicotine feeling, neuroprotection agent and the like.
The existing method for purifying and analyzing the cembratriene diol has various characteristics, but has the defects of residual derivatization reagent, low solvent recovery rate, complicated pretreatment and the like. Organic solvent extraction is used as a traditional separation method, and publication number: the patent publication of CN105001052A discloses a method for extracting cembrane di-mushroom from tobacco inflorescence, which comprises the steps of leaching by using an organic solvent, concentrating to obtain crude extract, removing higher alkane from the crude extract by using a solvent precipitation method, removing pigment by using activated carbon to obtain tobacco diterpene extract, and separating the obtained extract by using silica gel column chromatography to obtain two diterpene compounds. Publication No.: the CN102206138A discloses a method for separating and purifying two aroma precursors in tobacco, which adopts the steps of leaching, liquid-liquid extraction, normal phase silica gel column chromatography, normal Xiang Qingji column chromatography, reversed phase C18 column chromatography and the like to obtain the two aroma precursors. The existing traditional purification and separation methods have stable separation effect, but the separation efficiency still needs to be improved, and the development of new efficient separation and purification methods is the development direction of future separation.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a method for purifying cembratriene glycol in tobacco leaves by using multi-dimensional preparation liquid chromatography.
The invention provides a method for purifying cembratriene glycol in tobacco leaves by using multi-dimensional preparation liquid chromatography, which comprises the following steps:
A1 Adding a solvent into the tobacco leaf sample for leaching to obtain an extracting solution;
a2 Purifying and eluting the extract obtained in the step A1) by a first dimension liquid chromatographic column, purifying and eluting by a second dimension liquid chromatographic column, capturing and eluting by a capturing column array, and then performing third dimension liquid chromatographic column fraction and eluting to provide purified alpha-cembratriene glycol and beta-cembratriene glycol.
Preferably, in the step A1), the tobacco leaf sample is crushed and sieved tobacco leaf powder; the aperture of a sieving screen of the tobacco leaf sample is less than or equal to 300 meshes;
Preferably, in the step A1), the ratio of the added mass of the tobacco leaf sample to the added volume of the solvent is 100 g/1000 mL; preferably, in step A1), the solvent is an aqueous 85-95% ethanol solution;
Preferably, in step A1), the number of leaches is 2-4, preferably 3;
Preferably, in step A1), the leaching time is 9-11h, preferably 10h;
Preferably, in the step A1), filtering to remove residues after the leaching, combining leaching solutions, and recovering the solvent under reduced pressure; preferably, in step A2), the first dimension liquid chromatography column purification conditions are: the chromatographic column is LSI-100 chromatographic column with diameter of 20-800mm;
The mobile phase is water;
the running pressure of the chromatographic column is 1-10MPa;
preferably, in step A2), the second dimension liquid chromatography column purification conditions are: the chromatographic column is C18 chromatographic column with diameter of 20-800mm;
the running pressure of the chromatographic column is 1-10MPa;
the flow rate of the mobile phase is 30-50mL/min, preferably 40mL/min;
the detection wavelength is 210nm; the mobile phase is ethanol-water, the mobile phase A is ethanol, and the mobile phase B is water;
the running time is 80min; gradient elution.
The specific procedure of the gradient elution is as follows: 0 to 60min, phase A: the volume ratio of the phase B is 10:90-80:20, a step of; 60-80 min, phase A: the volume ratio of the phase B is 80:20-10:90.
Preferably, in step A2), the conditions of trapping and eluting are as follows: the particle size of the filler of the collecting column array collecting column is 10um-200um; the trapping column array trapping column is a C8 chromatographic column; the diameter of the trapping column array trapping column is 20-800mm;
the operation pressure of the trapping column is 1-10MPa;
The number of the trapping columns is 9;
Preferably, in step A2), the third dimensional liquid chromatography column purification conditions are: the chromatographic column is cyano chromatographic column with diameter of 20-800mm;
the running pressure of the chromatographic column is 1-10MPa;
the flow rate of the mobile phase is 30-50mL/min, preferably 40mL/min;
the detection wavelength is 210nm; the mobile phase is ethanol-isopropanol, wherein the mobile phase A is ethanol, and the mobile phase B is isopropanol; the running time is 40min; gradient elution;
The specific procedure of the gradient elution is as follows: 0-40 min, phase A: the volume ratio of the phase B is 95:5-90:10.
Preferably, in step A2), the conditions for trapping and eluting by the trapping column array are as follows: the particle size of the filler of the trapping column is 10-200 um; the trapping column is a C8 chromatographic column;
the diameter of the trapping column is 20-800mm;
the operation pressure of the trapping column is 1-10MPa;
The number of the trapping columns is 9;
Preferably, the first dimension chromatographic column is eluted by using a gel permeation mode, and the elution volume of the target component is 5-20 times of the column volume of the first dimension chromatographic column.
Preferably, the second-dimension chromatographic column is eluted by using a reverse phase mode, and the elution volume of the target component is 1-15 times of the column volume of the second-dimension chromatographic column.
Preferably, the volume of the chromatographic column of the trapping column array for capturing the target fraction is 3-20 times of the volume of the trapping column.
Preferably, the third dimension chromatographic column is eluted using a non-aqueous reverse phase mode, and the elution volume of the target component is 1 to 10 times the column volume.
Preferably, the volume ratio of the ethanol to the water solution is 10:90-95:5.
Preferably, the first trapping column captures an elution volume of 5.0BV to 6.1BV of the second dimension chromatographic column;
the second trapping column captures the elution volume of the second dimension chromatographic column from 6.3BV to 6.8 BV;
The third trapping column captures the elution volume of the second dimension chromatographic column from 6.9BV to 7.9 BV;
The fourth trapping column captures the elution volume of 7.9BV-8.6BV of the second dimension chromatographic column;
The fifth trapping column captures the elution volume of the second dimension chromatographic column from 9.0BV to 10.2 BV;
the sixth trapping column captures the elution volume of the second dimension chromatographic column from 10.2BV to 13.6 BV.
Preferably, the volume ratio of the ethanol-isopropanol solution is 95:5-50:50.
The invention has the following beneficial effects:
the method utilizes multidimensional chromatography, and separates the cembratriene diol in the tobacco in a multidimensional and high-efficiency manner. The cembratriene diol is a compound with relatively poor thermal stability, and the multidimensional chromatographic separation process is not contacted with the external environment, so that the cembratriene diol is more suitable for separating substances with poor thermal stability; the detection of multiple detectors eliminates interference and the result is reliable; complex pretreatment and intermediate treatment steps are not needed, the reagent consumption is reduced, and the novel concept of safety and environmental protection is realized; the whole separation process is monitored by an upper computer, so that automatic production is realized, and the separation process is not required to be manually interfered; the purity of the purified cembratriene diol is more than 99 percent, the recovery rate is high, the process is simple, the mass production is easy, and the method has important significance for separating the cembratriene diol.
Drawings
FIG. 1 is a schematic diagram of a multi-dimensional preparative liquid chromatography purification device for cembratriene diol in tobacco leaves;
FIG. 2 is a graph showing a first dimension of the separation of cembratriene diol from the multi-dimension preparation of liquid chromatography purified tobacco leaves according to the present invention;
FIG. 3 is a second dimension of the spectrum of the cembratriene diol in the multi-dimension preparation liquid chromatography purified tobacco leaves according to the present invention;
FIG. 4 is a third dimension of the spectrum of the cembratriene diol in the multi-dimensional preparative liquid chromatography purified tobacco leaves according to the invention (peak 1: alpha-cembratriene diol; peak 2: beta-cembratriene diol).
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
It should be understood that the process equipment or devices not specifically identified in the examples below are all conventional in the art.
As shown in figure 1, the device for purifying the cembratriene glycol in the tobacco leaves by using the multi-dimensional preparation liquid chromatography comprises a first pump system, a first switching valve, a second switching valve, a third switching valve, a first detector, a fourth switching valve, a trapping column array, a fifth switching valve, a sixth switching valve, a second detector and an automatic collector which are sequentially connected; the first switching valve is connected with a sample injection pump system, the second switching valve is connected with a first dimension chromatographic column system, the sixth switching valve is connected with a third dimension chromatographic column, and the automatic collector is externally connected with a waste liquid pool for collecting waste liquid;
The first switching valve, the second switching valve, the third switching valve and the sixth switching valve are six-way valves, 6 connecting sites are formed in total in 1-6 numbers, and the two working states A and B are formed, when the switching valve is placed in the position A, the number 1 bit of the switching valve is connected with the number 2 bit, the number 3 bit is connected with the number 4 bit, and the number 5 bit is connected with the number 6 bit; when the switching valve is arranged at the position B, the number 1 bit of the switching valve is connected with the number 6 bit, the number 2 bit is connected with the number 3 bit, and the number 4 bit is connected with the number 5 bit;
The fourth switching valve and the fifth switching valve are ten-position selection valves, each of which has 11 connection points 1-11, and has ten working states of A2, A3, A4, A5, A6, A7, A8, A9, A10 and A11, when the switching valve is placed at the position A2, the switching valve 1 is connected with the No. 2 phase, when the switching valve is placed at the position A3, the switching valve 1 is connected with the No. 3 phase, when the switching valve is placed at the position A4, the switching valve 1 is connected with the No. 4 phase, when the switching valve is placed at the position A5, the switching valve 1 is connected with the No. 5 phase, when the switching valve is placed at the position A6, the switching valve 1 is connected with the No. 7 phase, when the switching valve is placed at the position A8, the switching valve 1 is connected with the No. 8 phase, when the switching valve is placed at the position A9, the switching valve 1 is connected with the No. 9, when the switching valve is placed at the position A10, the switching valve 1 is connected with the No. 11, and when the switching valve is placed at the position A6;
The trapping column array comprises a first trapping column, a second trapping column, a third trapping column, a fourth trapping column, a fifth trapping column, a sixth trapping column, a seventh trapping column, an eighth trapping column, a ninth trapping column and a bypass;
The outlet end of the first pump system is connected with the first switching valve No. 1 through a pipeline, the first switching valve No. 2 is connected with the second switching valve No. 1 through a pipeline, and the first switching valve No. 3 is connected with the outlet end of the sample injection pump system through a pipeline;
The second switching valve No. 2 is connected to the inlet end of the first dimension chromatographic column through a pipeline, the second switching valve No. 3 is connected with the second switching valve No.5 through a pipeline, the second switching valve No. 4 is connected with the outlet end of the first dimension chromatographic column through a pipeline, and the second switching valve No.6 is connected to the third switching valve No. 1 through a pipeline;
The third switching valve No.2 is connected to the inlet end of the second-dimension chromatographic column through a pipeline, the third switching valve No. 3 is connected to the third switching valve No. 5 through a pipeline, the third switching valve No. 4 is connected to the outlet end of the second-dimension chromatographic column through a pipeline, and the third switching valve No. 6 is connected to the inlet end of the first detector through a pipeline; the outlet end of the first detector is connected to the fourth switching valve No.1 through a pipeline.
The fourth switching valve No. 2 is connected with the inlet end of the first trapping column through a pipeline, the fourth switching valve No. 3 is connected with the inlet end of the second trapping column through a pipeline, the fourth switching valve No. 4 is connected with the inlet end of the third trapping column through a pipeline, the fourth switching valve No. 5 is connected with the inlet end of the fourth trapping column through a pipeline, the fourth switching valve No. 6 is connected with the inlet end of the fifth trapping column through a pipeline, the fourth switching valve No. 7 is connected with the inlet end of the sixth trapping column through a pipeline, the fourth switching valve No. 8 is connected with the inlet end of the seventh trapping column through a pipeline, the fourth switching valve No. 9 is connected with the inlet end of the eighth trapping column through a pipeline, the fourth switching valve No. 10 is connected with the inlet end of the ninth trapping column through a pipeline, and the fourth switching valve No. 11 is connected with the fifth switching valve No. 11.
The fifth switching valve No. 1 is connected with the sixth switching valve No. 1 through a pipeline, the fifth switching valve No. 2 is connected with the first trapping column outlet end through a pipeline, the fifth switching valve No. 3 is connected with the second trapping column outlet end through a pipeline, the fifth switching valve No. 4 is connected with the third trapping column outlet end through a pipeline, the fifth switching valve No. 5 is connected with the fourth trapping column outlet end through a pipeline, the fifth switching valve No. 6 is connected with the fifth trapping column outlet end through a pipeline, the fifth switching valve No. 7 is connected with the sixth trapping column outlet end through a pipeline, the fifth switching valve No. 8 is connected with the seventh trapping column outlet end through a pipeline, the fifth switching valve No. 9 is connected with the eighth trapping column outlet end through a pipeline, and the fifth switching valve No. 10 is connected with the ninth trapping column outlet end through a pipeline;
the sixth switching valve No. 2 is connected with the inlet end of the third dimension chromatographic column through a pipeline, the sixth switching valve No. 3 is connected with the sixth switching valve No. 5 through a pipeline, the sixth switching valve No. 4 is connected with the outlet end of the third dimension chromatographic column through a pipeline, the sixth switching valve No. 5 is connected with the sixth switching valve No. 3 through a pipeline, the sixth switching valve No. 6 is connected with the inlet end of the second detector through a pipeline, the outlet end of the second detector is connected with the inlet end of the collector through a pipeline, the outlet end of the collector is connected to a waste liquid pond through a pipeline,
The collector is used for collecting the target component;
in the above-mentioned multi-dimensional liquid chromatography device for purifying cembratriene diol, the first dimension chromatographic column is an LSI-100 chromatographic column, the second dimension chromatographic column is a C18 chromatographic column, and the third dimension chromatographic column is a cyano chromatographic column.
In the above-described multi-dimensional liquid chromatography apparatus for purifying cembratriene diol, the array of trapping columns includes at least two trapping columns connected in parallel and a bypass; wherein the trapping column is a C8 chromatographic column.
The first dimension chromatographic column, the second dimension chromatographic column, the third dimension chromatographic column and the collecting column can be glass column or stainless steel column with diameter of 20-800mm, wherein the filler particle diameter is 10-200 μm, and the operating pressure is 1-10MPa.
The number of the chromatographic columns, the trapping columns and the number of the switching valves can be increased or decreased according to actual conditions.
Based on any one of the above multi-dimensional liquid chromatography devices, the invention also provides a method for purifying cembratriene diol, which comprises the following steps:
1) First dimension preparative liquid chromatography purification
Loading the tobacco leaf extract to a first-dimension chromatographic column, and eluting the first-dimension chromatographic column by taking water as flow; when the first dimension detector detects the target fraction eluted on the first dimension chromatographic column, all the target fractions are captured by using the second dimension chromatographic column by switching the chromatographic column switching device; enriching the target fraction into a second-dimension chromatographic column for second-dimension chromatographic separation; the first dimension chromatographic column is an LSI-100 chromatographic column with the specification of 30mm by 20mm; the volume ratio of the water is 100%, the elution volume of the target fraction is 5-20 times of the volume of the first dimension chromatographic column, and the first detector is an ultraviolet-visible light detector.
2) Second dimension preparative liquid chromatography purification
Eluting with ethanol-water as flow relative to the second dimension chromatographic column; when the second detector detects the target fraction eluted on the second-dimension chromatographic column, the trapping column switching device is switched to sequentially use the trapping column to capture different target fractions, and the target fractions are enriched into the trapping column array for third-dimension chromatographic separation; the second dimension chromatographic column is a C18-10um-B chromatographic column, the specification is 30 mm, the volume ratio of ethanol to water is 10:90-95:5, and the elution volume of the target fraction is 1-15 times of the volume of the second dimension chromatographic column. The trapping column array comprises 9 trapping columns, the first trapping column to the ninth trapping column are C8-10um chromatographic columns with the specification of 20mm, the volume of the trapping column trapping target fraction is 3-20 times of the volume of the trapping column, and the second detector is a differential detector.
3) Third dimension preparative liquid chromatography purification
Sequentially loading target fractions in an elution trapping column array to a third-dimensional chromatographic column for three-dimensional purification by taking ethanol-isopropanol as a mobile phase, and collecting a third-dimensional target fraction eluent by a fraction collector when the second detector detects the corresponding target fraction in the elution state of the third-dimensional chromatographic column; the third dimension chromatographic column is a cyano CN-10um chromatographic column with the specification of 30 mm, the volume ratio of ethanol to isopropanol is 95:5-50:50, and the elution volume of the target fraction is 1-10 times of the volume of the second dimension chromatographic column.
4) And drying the third-dimension target fraction eluent to obtain the product.
The specific purification steps of the invention are as follows:
Step 1, a first switching valve is positioned at a position B; the second switching valve is positioned at the A position; the third switching valve is positioned at the position B; the fourth switching valve and the fifth switching valve are in the A11 position; the sixth switching valve is at the position B; the first dimension chromatographic column is in a loading state. The sample injection pump system conveys an initial sample, the initial sample enters the first switching valve 2 from the first switching valve 2 into the second switching valve 1, the sample enters the second switching valve 4 from the first switching valve 4 into the second switching valve 6 into the third switching valve 1 from the second switching valve 4 into the second switching valve 6 into the first detector, the sample enters the fourth switching valve 1 from the third switching valve 1 into the third switching valve 6 into the first detector, the sample enters the fifth switching valve 11 from the fourth switching valve 1 into the fourth switching valve 11 from the fourth switching valve 11 into the sixth switching valve 1 from the sixth switching valve 1 into the sixth switching valve 6 into the second detector, and the sample enters the collector through the second detector and flows into the liquid pool.
Step 2, the first switching valve is positioned at the position A; the second switching valve is positioned at the A position; the third switching valve is positioned at the A position; the fourth switching valve and the fifth switching valve are in the A11 position; the sixth switching valve is at the position B; the first dimension chromatographic column is in an elution state, and the second dimension chromatographic column is in a loading state. The first pump system delivers water as the mobile phase to elute the first dimension chromatography column and maintains this state until the first dimension chromatography column is eluted to a column volume.
Step 3, the first switching valve is positioned at the position A; the second switching valve is at the B position; the third switching valve is positioned at the A position; the fourth switching valve and the fifth switching valve are in the A11 position; the sixth switching valve is at the position B; the second dimension chromatographic column is in a pre-eluting impurity state. The first pump system conveys the ethanol-water mobile phase, the ethanol-water mobile phase enters the second switching valve No. 1 from the first switching valve No. 1 to the first switching valve No. 2, the third switching valve No. 1 from the second switching valve No. 1 to the second switching valve No. 6, the third switching valve No. 1 to the third switching valve No. 2, the second dimension chromatographic column, the third switching valve No. 4 to the third switching valve No. 6, the first detector, the fourth switching valve No. 1, the fifth switching valve No. 11 to the fifth switching valve No. 11, the sixth switching valve No. 1 to the sixth switching valve No. 6, the second waste detector, the second detector, the collector and the liquid pool.
Step 4, the first switching valve is positioned at the position A; the second switching valve is at the B position; the third switching valve is positioned at the A position; the fourth switching valve and the fifth switching valve are in the A2 position; the sixth switching valve is at the position B; the second dimension chromatography column is in an eluted state. The first pump system conveys the ethanol-water mobile phase, the second dimension chromatographic column is eluted, the target object is enriched by the first trapping column through the first detector, and the waste liquid enters the waste liquid pool.
Step 5, the first switching valve is positioned at the position A; the second switching valve is at the B position; the third switching valve is positioned at the A position; the fourth switching valve and the fifth switching valve are in the A11 position; the sixth switching valve is at the position B; the second dimension chromatographic column is in a post-eluting impurity state. The first pump system delivers the ethanol-water mobile phase, elutes the second dimension chromatographic column, passes through the first detector, and the waste liquid enters the waste liquid pool.
And 6, repeating the steps 3-5, switching the fourth switching valve and the fifth switching valve to different states of A2-A10, and respectively trapping different target components by the first trapping column to the ninth trapping column. Repeating the steps 1-5, loading and eluting for multiple times, enriching micro-components on the trapping column, and carrying out third-dimension separation after enriching a certain amount of target components.
Step 7, the first switching valve is positioned at the position A; the second switching valve is at the B position; the third switching valve is positioned at the position B; the fourth switching valve and the fifth switching valve are in the A2 position; the sixth switching valve is at the A position; the third dimension chromatographic column is in a loading state. The first pump system conveys the ethanol-water mobile phase, the ethanol-water mobile phase enters the second switching valve 1 position from the first switching valve 1 position to the first switching valve 2 position, the third switching valve 1 position from the second switching valve 1 position to the second switching valve 6 position, the first detector enters the fourth switching valve 1 position from the third switching valve 6 position, the first trapping column enters the first trapping column from the fourth switching valve 1 position to the fourth switching valve 2 position, the fifth switching valve 2 position to the fifth switching valve 1 position, the sixth switching valve 1 position from the fifth switching valve 2 position to the sixth switching valve 2 position, the third three-dimensional chromatographic column enters the sixth switching valve 4 position from the sixth switching valve 4 position to the sixth switching valve 6 position, the second detector enters the second liquid pool from the sixth switching valve 4 position to the sixth switching valve 6 position, and the second detector enters the waste liquid pool from the second detector.
Step 8, the first switching valve is positioned at the position A; the second switching valve is at the B position; the third switching valve is positioned at the position B; the fourth switching valve and the fifth switching valve are in the A2 position; the sixth switching valve is at the A position; the third dimension chromatographic column is in a pre-eluting impurity state. The first pump system conveys the ethanol-water mobile phase, the third chromatographic column is eluted, the second detector is passed through, and the waste liquid enters the waste liquid pool.
Step 9, the first switching valve is positioned at the A position; the second switching valve is at the B position; the third switching valve is positioned at the position B; the fourth switching valve and the fifth switching valve are in the A11 position; the sixth switching valve is at the A position; the third dimension chromatography column is in an eluted state. The first pump system conveys the ethanol-water mobile phase, the third chromatographic column is eluted, the target object is collected by the collector through the second detector, and the waste liquid enters the waste liquid pool.
Step 10, the first switching valve is positioned at the A position; the second switching valve is at the B position; the third switching valve is positioned at the position B; the fourth switching valve and the fifth switching valve are in the A11 position; the sixth switching valve is at the A position; the third dimension chromatographic column is in a eluting impurity state. The first pump system conveys the ethanol-water mobile phase, the third chromatographic column is eluted, the second detector is passed through, and the waste liquid enters the waste liquid pool.
And 11, repeating the steps 7-10, switching the fourth switching valve and the fifth switching valve to different states of A2-A10, and loading different target substances trapped by the first trapping column to the ninth trapping column to the third chromatographic column for third-dimension purification.
And 12, repeating the steps 2-11. Thus, a complete process of sample injection, enrichment, elution and regeneration is completed.
Example 1
Weighing 100g of crushed tobacco leaf powder below 300 meshes, adding 1000mL of 90% ethanol solution, leaching for 3 times, 10 hours each time, filtering to remove residues, mixing the extracting solutions, and recovering part of ethanol solvent under reduced pressure to obtain crude extracting solution;
Loading the obtained crude extract to a first dimension chromatographic column which is an LSI-100 chromatographic column with the height of 30 x 20mm, and eluting the 15 th column volume by taking water as a mobile phase; the switching valve enables eluent with the volume of 5-15 times of column to flow into a second-dimension chromatographic column, the target component is loaded on the second-dimension chromatographic column, the second-dimension chromatographic column is 30 x 250mm C18-10um-B chromatographic column, ethanol-water is taken as a mobile phase, the initial ethanol-water ratio is 10:90, and the ratio is 80 in 60 minutes: the ratio at 20, 80 minutes is 90:10, the flow rate is 40mL/min, and the detection wavelength of the first detector is 210nm; the trap column traps the target object in response to the first detector signal by switching the valve.
The first trapping column captures a second fraction having an elution volume of 5.0BV to 6.1BV on a second dimension chromatography column; the second trapping column captures a third fraction having an elution volume of 6.3BV to 6.8BV on the second dimension chromatography column; the third trapping column captures a fourth fraction having an elution volume of 6.9BV to 7.9BV on the second dimension chromatography column; the fourth trapping column captures a fifth fraction which is an elution volume of 7.9BV to 8.6BV on the second dimension chromatography column; the fifth trapping column captures a sixth fraction having an elution volume of 9.0BV to 10.2BV in the second dimension chromatography column; the sixth trapping column captures a seventh fraction (containing cembratriene diol) which has an elution volume of 10.2BV to 13.6BV on the second dimension column.
The switching valve is used for connecting the trapping column with a third-dimensional chromatographic column, eluting the trapping column, and loading the sample to the third-dimensional chromatographic column, wherein the third-dimensional chromatographic column is a 30 x 250mm CN-10um chromatographic column. Loading the fraction trapped by the sixth trapping column to a third chromatographic column for separation, wherein the ethanol-isopropanol is taken as a mobile phase, and the initial ethanol-isopropanol ratio is 95: the ratio at 5, 40 minutes is 90:10, the flow rate is 40mL/min, and the detection wavelength is 210nm; according to the second detector signal, the switching valve and the fraction collector collect the target, the elution volume of the first target (alpha-cembratriene glycol) is 2.25BV-2.49BV, the elution volume of the second target (beta-cembratriene glycol) is 4.73BV-4.97BV, the target fraction is freeze-dried, and the detection results are shown in Table 1:
Table 1 table of product names, purity and yield in example 1
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (10)
1. A method for purifying cembratriene diol in tobacco leaves by multi-dimensional preparation liquid chromatography, which is characterized by comprising the following steps:
A1 Adding a solvent into the tobacco leaf sample for leaching to obtain an extracting solution;
a2 Purifying and eluting the extract obtained in the step A1) by a first dimension liquid chromatographic column, purifying and eluting by a second dimension liquid chromatographic column, capturing and eluting by a capturing column array, and then performing third dimension liquid chromatographic column fraction and eluting to provide purified alpha-cembratriene glycol and beta-cembratriene glycol;
In the step A2), the purification conditions of the first dimension liquid chromatographic column are as follows: the chromatographic column is LSI-100 chromatographic column with diameter of 20-800mm;
The mobile phase is water;
the running pressure of the chromatographic column is 1-10MPa;
in the step A2), the second dimension liquid chromatography column purifying conditions are as follows: the chromatographic column is C18 chromatographic column with diameter of 20-
800mm；
The running pressure of the chromatographic column is 1-10MPa;
The flow rate of the mobile phase is 30-50mL/min;
the detection wavelength is 210nm; the mobile phase is ethanol-water, the mobile phase A is ethanol, and the mobile phase B is water;
The running time is 80min; gradient elution;
the specific procedure of the gradient elution is as follows: 0 to 60min, phase A: the volume ratio of the phase B is 10:90-80:20, a step of;
60-80 min, phase A: the volume ratio of the phase B is 80:20-10:90;
in the step A2), the conditions of trapping and eluting are as follows: the particle size of the filler of the trapping column array trapping column is 10 mu m
200Um; the trapping column array trapping column is a C8 chromatographic column; the diameter of the trapping column array trapping column is 20-800mm;
the operation pressure of the trapping column is 1-10MPa;
The number of the trapping columns is 9;
In the step A2), the third dimension liquid chromatography column purifying conditions are as follows: the chromatographic column is cyano chromatographic column with diameter of 20-800mm;
the running pressure of the chromatographic column is 1-10MPa;
The flow rate of the mobile phase is 30-50mL/min;
The detection wavelength is 210nm; the mobile phase is ethanol-isopropanol, wherein the mobile phase A is ethanol, and the mobile phase B is isopropanol;
the running time is 40min; gradient elution;
the specific procedure of the gradient elution is as follows: 0-40 min, phase A: the volume ratio of the phase B is 95:5-90:10;
in the step A2), the conditions of trapping and eluting through the trapping column array are as follows: the particle size of the filler of the trapping column is 10 mu m
200Um; the trapping column is a C8 chromatographic column;
the diameter of the trapping column is 20-800mm;
the operation pressure of the trapping column is 1-10MPa;
The number of trapping columns is 9.
2. The method for purifying cembratriene glycol in tobacco leaves by multi-dimensional preparation liquid chromatography according to claim 1, wherein the method comprises at least one of the following technical characteristics:
b1 In the step A1), the tobacco leaf sample is crushed and sieved tobacco leaf powder; the aperture of a sieving screen of the tobacco leaf sample is less than or equal to 300 meshes;
b2 In the step A1), the ratio of the added mass of the tobacco leaf sample to the added volume of the solvent is 100 g/1000 mL;
B3 In the step A1), the solvent is 85-95% ethanol water solution;
b4 In step A1), the leaching times are 2-4 times;
b5 In step A1), the leaching time is 9-11h;
B6 In the step A1), filtering to remove residues after the leaching, combining leaching solutions, and recovering and removing the solvent under reduced pressure;
b7 In the step A2), the purification conditions of the second-dimension liquid chromatographic column are as follows: the chromatographic column is a C18 chromatographic column with the diameter of 30mm;
the flow rate of the mobile phase is 40mL/min;
b8 In the step A2), the purification conditions of the third three-dimensional liquid chromatographic column are as follows: the chromatographic column is a cyano chromatographic column with the diameter of 30mm;
the mobile phase flow rate was 40mL/min.
3. The method for purifying cembratriene glycol in tobacco leaves by multi-dimensional preparation liquid chromatography according to claim 2, wherein the method comprises at least one of the following technical characteristics:
B41 Step B4), the leaching times are 3 times;
B51 In step B5), the leaching time is 10h.
4. The method for purifying cembratriene glycol in tobacco leaves by multi-dimensional preparation liquid chromatography according to claim 1, wherein the first dimension chromatographic column is eluted by using a gel permeation mode, and the elution volume of the target component is 5-20 times of the volume of the first dimension chromatographic column.
5. The method for purifying cembratriene glycol in tobacco leaves by multi-dimensional preparation liquid chromatography according to claim 1, wherein the second-dimension chromatographic column is eluted by a reverse phase mode, and the elution volume of the target component is 1-15 times of the volume of the second-dimension chromatographic column.
6. The method for purifying cembratriene glycol in tobacco by multi-dimensional preparation liquid chromatography according to claim 1, wherein the volume of the chromatographic column capturing target fraction of the trapping column array is 3-20 times of the volume of the trapping column.
7. The method for purifying cembratriene glycol in tobacco leaves by multi-dimensional preparation liquid chromatography according to claim 1, wherein the third chromatographic column is eluted by a non-aqueous reversed phase mode, and the elution volume of the target component is 1 to 10 times the column volume.
8. The method for purifying cembratriene glycol in tobacco leaves by multi-dimensional preparation liquid chromatography according to claim 1, wherein the volume ratio of the ethanol-water solution is 10:90-95:5.
9. The method for purifying cembratriene glycol in tobacco leaves by multi-dimensional preparation liquid chromatography according to claim 1, wherein the first trapping column captures an elution volume of 5.0BV-6.1BV of the second dimension chromatographic column;
the second trapping column captures the elution volume of the second dimension chromatographic column from 6.3BV to 6.8 BV;
The third trapping column captures the elution volume of the second dimension chromatographic column from 6.9BV to 7.9 BV;
The fourth trapping column captures the elution volume of 7.9BV-8.6BV of the second dimension chromatographic column;
The fifth trapping column captures the elution volume of the second dimension chromatographic column from 9.0BV to 10.2 BV;
the sixth trapping column captures the elution volume of the second dimension chromatographic column from 10.2BV to 13.6 BV.
10. The method for purifying cembratriene glycol in tobacco leaves by multi-dimensional preparation liquid chromatography according to claim 1, wherein the volume ratio of the ethanol-isopropanol solution is 95:5-50:50.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102206138A (en) * | 2010-12-30 | 2011-10-05 | 上海烟草集团有限责任公司 | Method for separating and purifying two fragrance precursors from tobacco |
| CN103561595A (en) * | 2011-04-27 | 2014-02-05 | R·J·雷诺兹烟草公司 | Tobacco-derived components and materials |
| CN105001052A (en) * | 2015-07-03 | 2015-10-28 | 中国农业科学院烟草研究所 | Method used for extracting cembrenediols from tobacco inflorescence |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206138A (en) * | 2010-12-30 | 2011-10-05 | 上海烟草集团有限责任公司 | Method for separating and purifying two fragrance precursors from tobacco |
| CN103561595A (en) * | 2011-04-27 | 2014-02-05 | R·J·雷诺兹烟草公司 | Tobacco-derived components and materials |
| CN110140993A (en) * | 2011-04-27 | 2019-08-20 | R·J·雷诺兹烟草公司 | Component and material from tobacco |
| CN105001052A (en) * | 2015-07-03 | 2015-10-28 | 中国农业科学院烟草研究所 | Method used for extracting cembrenediols from tobacco inflorescence |
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